Compositions and methods for producing vascular occlusion using a solid-phase platelet binding agent

ABSTRACT

The present invention relates generally to methods and compositions for targeting and delivering solid-phase platelet-dependent vascular occlusion agents. In particular, particles or coils or stents coated with platelet binding agents are directed to target vasculature, such as the vasculature of solid tumor masses or AV-malformations or aneurysms or endoleaks; the solid-phase agent then binds and activates platelets, which in turn bind and activate other platelets. This process results in the rapid formation of a platelet-mediated thrombus about the solid-phase agent causing vessel occlusion.

This application is a continuation of U.S. application Ser. No.10/241,717 filed Sep. 12, 2002, which is a non-provisional of U.S.application Ser. No. 60/318,339 filed Sep. 12, 2001. These patentapplications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention is directed to compositions and methods forproducing a therapeutic benefit by producing vascular occlusion usingplatelet activation as the initiating event. Compositions and methods ofthe invention involve delivering a solid-phase platelet-binding agent toa target site, causing platelets to bind and activate thereby forming alocalized thrombus. Occlusion of the vasculature of the target tissue bythe localized thrombus results in deprivation of essential oxygen andnutrients, in turn leading to tissue regression and ultimately tissuedeath.

DESCRIPTION OF RELATED ART

Platelets function in the body to limit blood loss in the event ofvascular damage. Normally, platelets circulate throughout the body withother cellular components of blood, bathed in a mixture of variousplasma proteins, many of which play key roles in the clotting process.Upon exposure of vascular sub-endothelium, a complex series of eventsoccurs to limit the loss of blood from the damaged vessel. Circulatingplatelets contacting components of the exposed sub-endothelium: 1) bindand adhere, 2) spread across the exposed surface, 3) activate asevidenced by release of granule contents, 4) aggregate and recruit othercirculating platelets from the blood stream, and 5) form an efficientplug, clot, and/or thrombus stemming the flow of blood from the vessel.

In response to vascular injury, such as atherosclerotic plaque rupturein a coronary vessel, circulating platelets are exposed to a variety ofmatrix elements that are prothrombotic. Platelets can strongly adhere totwo specific matrix components, collagen and von Willebrand factor. Atlow blood flow shear rates, adhesion to collagen predominates, whereasat higher shear rates—for example, those that would occur in stenosedvessels—the initial platelet adhesion is primarily mediated by bindingto von Willebrand factor. Adhesion to either collagen and/or vonWillebrand factor initiates signals leading to platelet activation,platelet spreading on the matrix, secretion of prothrombotic substancessuch as adenosine diphosphate (ADP) and thromboxane A₂, and upregulationof the adhesive function of GP IIb-IIIa, which can bind fibrinogen andvon Willebrand factor, resulting in platelet aggregate or thrombusformation.

In contrast to the coagulation cascade, a process defined in part by theconversion of fibrinogen to fibrin, platelets coalesce about the damagedarea and are held together by bridging molecules that bind to specificreceptors on the platelet surface. The initial bridging betweenplatelets and the sub-endothelium is dependent on the interactionbetween the glycoprotein Ib (GPIb) receptor on the surface of theplatelet and von Willebrand Factor (VWF) in the subendothelium (i.e.,immobilized VWF). This interaction in itself is unique, since normalplatelets circulating in the blood often contact soluble VWF, but arenot activated, nor do they bind to the soluble VWF. In vitroexperimentation has confirmed that immobilization of the soluble VWF toa surface facilitates binding and activation of platelets. Uponactivation of the platelet, an additional receptor, glycoproteinIIb/IIIa (GPIIb/IIIa), is altered enabling the binding of several plasmaproteins, thereby promoting platelet/platelet binding.

Hyperactive platelets can induce thrombus formation at inopportune timesresulting in reduced blood supply to various organs and tissues. A primeexample is thrombus formation induced by blood flowing through astenotic (narrowed) vessel supplying the heart. Reduction of the flow ofblood to the heart muscle leads to infarction and eventually heartattack (cardiac cell death). Cerebral ischemia (transient ischemicattack (TIA); stroke) occurs when an embolus or thrombus occludes bloodvessels feeding the brain.

Other pathological states exist that are caused by platelet activationas a result of an inappropriate antibody-mediated process.Heparin-induced thrombocytopenia (HIT) is characterized by a dramaticloss in platelet numbers and thrombus formation at sites of pre-existingpathology. From 1% to 5% of all patients receiving unfractionatedheparin as an anticoagulant to promote blood flow produce an antibodythat binds to heparin in complex with a platelet granule protein. Thebinding of the antibody to the heparin/protein complex on the surface ofthe platelet induces rapid platelet activation and localized thrombusformation. This in turn leads to infarction of the affected area.

Thrombosis is a well-described consequence of cancer. Controversy existsas to whether the presence of a hyper-coagulable state is predictive ofcancer. Many studies have been conducted demonstrating a prothrombotictendency with most neoplasia or neoplasms. It has been suggested thatthrombosis is the most frequent complication with patients with overtmalignant disease.

A key to the development of successful anti-tumor agents is the abilityto design agents that will selectively kill tumor cells, while exertingrelatively little, if any, untoward effects against normal tissues. Thisgoal has been elusive in that there are few qualitative differencesbetween neoplastic and normal tissues. Because of this, much researchover the years has focused on identifying tumor-specific “markerantigens” that can serve as immunological targets both for chemotherapyand diagnosis. Many tumor-specific or quasi-tumor-specific(tumor-associated) markers have been identified as tumor cell antigensthat can be recognized by specific antibodies.

Unfortunately, it is generally the case that tumor-specific antibodieswill not in and of themselves exert sufficient anti-tumor effects tomake them useful in cancer therapy. In contrast with their efficacy inlymphomas, immunotoxins have proven to be relatively ineffective in thetreatment of solid tumors such as carcinomas. The principal reason forthis is that solid tumors are generally impermeable to antibody-sizedmolecules: specific uptake values of less than 0.001% of the injecteddose per gram of tumor are not uncommon in human studies. Furthermore,antibodies that enter the tumor mass do not distribute evenly forseveral reasons. Firstly, the dense packing of tumor cells and fibroustumor stromas present a formidable physical barrier to macro-moleculartransport and combined with the absence of lymphatic drainage create anelevated interstitial pressure in the tumor core which reducesextravasation and fluid convection. Secondly, the distribution of bloodvessels in most tumors is disorganized and heterogeneous. As a resultsome tumor cells are separated by large distances from capillaries sothat the extravasating antibody must diffuse over a large volume inorder to reach and bind to remote tumor cells. Thirdly, all of theantibody entering the tumor may become absorbed in perivascular regionsby the first tumor cells encountered, leaving none to reach tumor cellsat more distant sites.

One approach to overcoming the deficiencies of targeting tumors withantibodies would be to target thrombus-inducing agents to thevasculature of the tumor rather than to the tumor.

The present inventors propose that this approach will provide severaladvantages over targeting tumor cells directly. Firstly, the targetcells are directly accessible to vascularly administered therapeuticagents permitting rapid localization of a high percentage of theinjected dose. Secondly, since each capillary provides oxygen andnutrients for thousands of cells in its surrounding cord of tumor, evenlimited damage to the tumor vasculature could produce an avalanche oftumor cell death.

The present invention is also directed to compositions and methods oftreating abnormal tissue growth, abnormal bleeding (during or aftersurgery, postpartum), ectopic pregnancy, placenta previa, placentaaccreta and uterine fibroids.

Under certain clinical situations, inhibition of blood flow to a tissuethrough occlusion of its associated vasculature is desirable. Examplesinclude hemorrhagic stroke, existence of saphenous vein side branches insaphenous bypass graft surgery, treatment of aortic aneurysm, correctionof vascular malformations, and treatment of solid tumors.

Vascular occlusion has been performed using a variety of techniques andmaterials including embolotherapy. Examples of embolotherapy include theuse of particles composed of a variety of materials including polyvinylalcohol (Boschetti, PCT WO0023054), acrylamide (Boschetti et al, U.S.Pat. No. 5,635,215; Boschetti et al, U.S. Pat. No. 5,648,100),polymethyl methacrylate (Lemperle, U.S. Pat. No. 5,344,452), physicalplugs composed of collagen (Conston et al, U.S. Pat. No. 5,456,693) andcoils (Mariant, U.S. Pat. No. 5,639,277). Embolotherapy involves thedelivery of these materials to the target vasculature by means of acatheter. Since the vasculature in any given area proceeds from largerarteries to arterioles to metarterioles to capillaries, each withprogressively smaller vessel diameters, the delivered material (embolus)continues to travel in the flowing blood until it becomes lodged in thesmaller blood vessels thereby impeding the flow of blood to thedependent tissue.

The present invention is novel and addresses unmet medical needs throughthe use of a solid-phase material, such as microparticles or coils orstents, coated with porous polylactide, polyglycolide orpolylactide-co-glycolide particles coated with mammalian collagen. Inthis way a therapeutic benefit may be achieved by delivering asolid-phase platelet-binding agent to a target site and initiatingefficient thrombus formation leading to occlusion of the associatedvasculature.

SUMMARY OF THE INVENTION

The present invention relates to therapeutic methods and compositionsfor targeting tissues and/or organs, and associated vasculature, whichare hyperplastic or neoplastic in nature, or which have arterio-venousmalformations, or which are hemorrhaging, using solid-phase agents thatinduce thrombus formation via localized platelet activation. Thecomposition comprises an agent for capturing platelets on a solid-phaseagent such as a coil or a stent or a particle. In some embodiments ofthe invention, the solid-phase agent both captures and activates theplatelets. The method utilizes localizing platelet collection andactivating the platelets on the solid-phase particle to producesubsequent thrombus formation, thereby limiting the blood supply to thetarget area, without inducing a generalized or systemic pro-thromboticstate.

Purposeful induction of thrombosis in a patient appears at first glanceto be counter-intuitive, since thrombosis is well known to contributesignificantly to patient morbidity and mortality. The present invention,solid-phase platelet-mediated occlusion, is based on the site-specificinduction of thrombosis utilizing the body's natural capacity to producea thrombus in response to porous polylactide, polyglycolide orpolylactide-co-glycolide particles coated with mammalian collagen orother locally acting platelet activation agents. The immobilizedcollagen, acting as a platelet binding agent, serves to anchor plateletsto the particles through two mechanisms: 1) directly, by binding to keyplatelet membrane receptors such as α2β1 and GPVI; and 2) indirectly, bycapturing circulating, autologous, von Willebrand factor (VWF), which inturns captures and activates platelets circulating in the blood streamthrough key platelet membrane receptors such GPIb and GPIIb/IIIa.Although VWF circulates in the blood stream in soluble form, it is notuntil the molecule is exposed as part of the subendothelium or binds toexposed collagen from the subendothelium that it is capable of capturingplatelets and inducing platelet activation.

Contact of the solid-phase platelet-binding agent with the blood from apatient (ex vivo) or in the blood stream (in vivo) induces plateletbinding and localized activation leading to accretion of platelets aboutthe solid-phase agent leading to thrombosis and cessation of blood flowto the tissue supplied by the occluded blood vessel(s). Cells, includingtumor cells or hyperplastic tissue, diminish or die as a result of lossof localized blood flow. This approach avoids systemic plateletactivation and thrombosis; relying on the fact that porous polylactide,polyglycolide or polylactide-co-glycolide particles coated withmammalian collagen binds to and activates circulating platelets. Thus,the methods and compositions of the present invention are an indirectmeans of treating a pathological condition, such as cancer, hyperplasticcells, excessive bleeding or arteriovenous (AV) malformations.

The present invention improves on existing methods for treating solidtumors, hyperplastic tissue, excessive bleeding and AV-malformations andany other disease or condition in which platelets (resting and/oractivated) may play a therapeutic role.

In a manner similar to an existing pathological condition (i.e.Heparin-Induced Thrombocytopenia [HIT]), localized platelet activationcan be enhanced by means of an Fc-mediated process by including orincorporating a human Fc fragment onto the solid-phase agent, or bydirecting select antibodies to the target area. Platelet activation inHIT syndrome results in localized thrombosis and cessation of blood flowto the affected area. This leads to death of the affected tissue.

The extent or degree of site-specific thrombosis can be controlled in avariety of ways. Inhibition of platelet activation through the use ofanti-platelet agents (e.g. GPIIb/IIIa inhibitors, aspirin, dipyridamole,etc.) decreases the propensity to induce a thrombus in a defined,titratable manner. Altering local blood flow, blood pressure and tissuetemperature can also serve as means of controlling local plateletactivation to a stimulus.

Typical vascularized tumors are the solid tumors, particularlycarcinomas, which require a rich vascular blood supply. Exemplary solidtumors to which the present invention is directed include, but are notlimited to, primary malignant tumors of the lung, breast, ovary,stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliarytract, colon, rectum, cervix, uterus, endometrium, kidney, bladder,prostate, thyroid, head and neck, melanomas, gliomas, neuroblastomas,neuroendocrine tumors, and the like. Other conditions to which thepresent invention is directed include, but are not limited to, secondary(metastatic) tumors of the above mentioned tumor types, cancer pain,AV-malformations, uterine fibroids, pelvic congestion, menorrhagia,varicoceles, hemoptysis, aneurysms, visceral artery aneurysms,pseudoaneurysms and endoleaks.

A preferred method of the invention includes preparing a coil or stentcoated with porous polylactide, polyglycolide orpolylactide-co-glycolide particles coated with mammalian collagen(PLGA/Collagen) and introducing the PLGA/Collagen-coated agent into thebloodstream of an animal, such as a human patient, an animal patient, ora test animal; the PLGA/Collagen is then delivered or collects at adesired target site. The coils or stents can be constructed of anysuitable material capable of retaining PLGA/Collagen either within thecoil or stent or on the surface of the coil or stent for an indefiniteor varying lengths of time.

A solution to the problem of the unrestrained growth of solid tumors isto attack the blood vessels in the tumor. This approach offers severaladvantages over methods that directly target tumor cells. Firstly, thetumor vessels are directly accessible to vascularly administeredtherapeutic agents, thus permitting rapid localization of highpercentage of the injected dose. Secondly, since each capillary providesoxygen and nutrients for thousands of cells in its surrounding cord oftumor even limited damage to the tumor vasculature could produceextensive tumor cell death. Finally, blood vessels are similar indifferent tumors, making it feasible to develop a single reagent fortreating numerous types of cancer.

DESCRIPTION OF THE DRAWINGS

Not Applicable

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for capturingplatelets at a predetermined site, activating the platelets, andharnessing the natural function of platelets to achieve a beneficialtherapeutic result. In accordance with the present invention, theplatelets may be circulating platelets or may be platelets obtained froman external source. In accordance with the present invention, plateletsmay be targeted to a specific site, and then the natural ability ofplatelets to induce thrombus formation may be used to interrupt,disrupt, or reduce blood flow at the site. Reduced blood flowconcomitantly reduces nutrient supply to a disease or condition agent,such as a tumor, so the size of the disease agent is diminished. It isclear that reducing the size of a tumor is an obvious therapeuticbenefit. In some instances reduction of the blood supply to a targetarea alleviates pain.

The present invention also includes targeting platelets to apre-determined tissue capable of being selectively targeted, e.g.,hyperplastic tissue, using a solid-phase agent capable of binding andactivating the platelets. In these embodiments of the invention,targeting refers to the solid phase containing a targeting moiety, e.g.,a ligand or the like, that specifically binds the pre-determined site ortissue. In other embodiments of the invention, targeting may includedelivering a composition of the present invention at or near a tumorsite, e.g., by catheter, stent, or coil. Activating the platelets at thepre-selected site causes a therapeutic benefit by reducing the nutrientsupply to the tissue or site.

The present invention provides compositions and methods for inducingthrombus formation by capturing platelets on the solid-phase agent,inducing activation of the platelets, and allowing a thrombus to form.Thrombus formation in the target vasculature reduces the blood supply tothe downstream tissue. By capturing platelets on aPLGA/Collagen-containing solid phase agent, the compositions and methodsof the present invention may be used to treat cancer, hyperplasia,uterine fibroids, pelvic congestion, menorrhagia, AV-malformations,neuro-embolism, varicoceles, hemoptysis, visceral artery aneurysms,arterial aneurysms, endoleaks, and the like. Furthermore, thecompositions and methods of the present invention provide a therapeuticbenefit to the recipient of the composition.

In a preferred embodiment of the invention, the PLGA/Collagen is ofmammalian origin. In a most preferred embodiment of the invention, thePLGA/Collagen is of human origin. In a further most preferred embodimentof the invention, the PLGA/Collagen is of bovine origin.

The PLGA/Collagen may be natural, synthetic, recombinant, or a peptidesequence conforming to a biologically active portion of PLGA/Collagen.In a further most preferred embodiment of the invention, thePLGA/Collagen is of recombinant origin.

The present invention also provides compositions that bind aplatelet-binding agent directly or indirectly through a spacer to thesolid phase, so long as the ability of the platelet-binding agent tobind platelets is not impaired. Spacer, as used herein, refers to agroup of inert or active molecules that physically separate the plateletbinding agent from the surface of the solid phase agent. Exemplaryspacers are described below. The direct binding can occur eithercovalently or non-covalently. Indirect binding can occur throughspacers, including but not limited to peptide spacer arms, antibodyspacers, antibody fragment spacers, fusion protein spacers orcarbohydrate spacers. These spacers normally act only as bridges betweenthe particle and the PLGA/Collagen; however, the spacers could also beused to alter the degree of platelet activation. For example, an Fccomponent could be used as a spacer, thereby effecting enhanced plateletactivation on and about the solid-phase agent. Coupling of PLGA/Collagento the solid phase agent can occur using methods known to those skilledin the art. Examples of coupling agents include but are not limited toglutaraldehyde and carbodiimide.

In a preferred embodiment of the invention, the positioning within thevascular system of mammals of compositions without an active targetingagent would be selected by blood flow directed positioning followingdelivery by means of a superselective microcatheter.

Compositions according to the present invention may also include atargeting agent or moiety capable of binding a target antigen or site onthe vascular endothelium or target tissue, thereby enabling localizationof the solid-phase agent to a selected site. Exemplary targeting agentsor moieties are well known to those skilled in the art, and include, butare not limited to antibodies, ligands, receptors, hormones, lectins,and cadherins, or portions or fragments thereof.

In a preferred embodiment of the invention, the targeting agent wouldinclude an antibody or antibody-like molecule with biotin, biotinmimetic and/or a peptide component. In a further preferred embodiment ofthe invention, the antibody or antibody-like molecules would be directedtoward a growth factor/receptor complex.

Compositions according to the invention may also include one or more ofthe following: one or more platelet binding modulators (e.g., inhibitorsor enhancers), one or more thrombus formation controllers or modulatorsor one or more complement cascade components.

Methods according to the invention may also include administering asolid-phase agent capable of binding platelets at a pre-determined site;may also include inducing activation of the captured platelets;administering a bifunctional binding agent having an antigenicdeterminant and a platelet binding site; controlling thrombus generationby altering the temperature of one or more compositions of theinvention, or by altering the temperature at the pre-selected site.

Methods according to the invention may further include one or more ofthe following: administering one or more platelet binding modulators,administering one or more thrombus formation modulators; administeringone or more complement cascade components; administering one or moreligands and/or anti-ligands for binding the solid phase to apre-determined site, and/or for binding a platelet binding moiety orcomponent to the solid phase.

The present invention also includes a kit which may contain but is notlimited to any or all of the following components including asolid-phase agent for targeting platelets to an endothelial membranecomponent: a binding agent for binding platelets; a ligand for bindingan endothelial membrane component; a ligand conjugate; an anti-ligandfor binding the ligand or the ligand conjugate; a platelet bindingmodulator (enhancer and/or inhibitor); a thrombus formation modulator; acomplement cascade component; a complement cascade component inducer;and a binding agent for binding platelets that includes an anti-ligand.The kit may include a bifunctional binding agent, and/or a bindingagent-ligand conjugate, and/or a platelet-binding agent-anti-ligandconjugate.

The compositions and methods of the present invention include anymechanism of delivering a composition to the pre-selected site,including but not limited to systemically, locally, orally, ortopically.

In accordance with some embodiments of the invention, binding agents areused to capture platelets at a predetermined site.

Definitions:

As used herein, a solid-phase agent refers to any solid materialsuitable for binding, containing, or retaining a platelet-binding agent.The platelet-binding agent may be attached to the solid-phase agent suchthat platelet binding activity is retained, e.g., at or within a targetsite. The solid phase agent may be a coil, stent, or particle, e.g., abead or the like, all of which are well known to those skilled in theart.

As used herein, a particle refers to a discrete portion or part of asolid-phase material capable of containing or retaining aplatelet-binding agent.

A preferred method of the invention includes preparing a particle coatedwith PLGA/Collagen of recombinant or mammalian origin and introducingthe PLGA/Collagen-coated particle into the bloodstream of an animal,such as a human patient, an animal patient, or a test animal.

As used herein, the term “particle” refers to any solid-phase materialcapable of binding platelets, either directly or indirectly (e.g.,through ligands). The particles can be homogenous or heterogeneous asrelated to size. Specifically, the particles can be of spherical(including ovoid) or irregular shape. The particles can be constructedof any suitable material capable of retaining collagen either within theparticle or on the surface of the particle for an indefinite or varyinglengths of time. Exemplary materials include polyvinyl alcohol (PVA),polystyrene, polycarbonate, polylactide, polyglycolide,lactide-glycolide copolymers, polycaprolactone, lactide-caprolactonecopolymers, polyhydroxybutyrate, polyalkylcyanoacrylates,polyanhydrides, polyorthoesters, albumin, gelatin, polysaccharides,dextrans, starches, methyl methacrylate, methacrylic acid, hydroxylalkylacrylates, hydroxylalkyl methacrylates, methylene glycol dimethacrylate,acrylamide, bisacrylamide, cellulose-based polymers, ethylene glycolpolymers and copolymers, oxyethylene and oxypropylene polymers,polyvinyl acetate, polyvinylpyrrolidone and polyvinylpyridine, magneticparticles, fluorescent particles, animal cells, plant cells,macro-aggregated and micro-aggregated albumin, denatured proteinaggregates and liposomes, used singly or in combination.

In the present invention the preferred solid phase material ispolylactide, polyglycolide or polylactide-co-glycolide. The solid phasematerials suitable for use in the present invention are well known tothose skilled in the art, and should not be limited to those exemplarymaterials recited above.

Exemplary materials for forming the stent or coil include, but are notlimited to: polyvinyl alcohol (PVA), polystyrene, polycarbonate,polylactide, polyglycolide, lactide-glycolide copolymers,polycaprolactone, lactide-caprolactone copolymers, polyhydroxybutyrate,polyalkylcyanoacrylates, polyanhydrides, polyorthoesters,polysaccharides, dextrans, starches, methyl methacrylate, methacrylicacid, hydroxylalkyl acrylates, hydroxylalkyl methacrylates, methyleneglycol dimethacrylate, acrylamide, bisacrylamide, cellulose-basedpolymers, ethylene glycol polymers and copolymers, oxyethylene andoxypropylene polymers, polyvinyl acetate, polyvinylpyrrolidone andpolyvinylpyridine; magnetic materials, fluorescent materials; gold,platinum, palladium, rhenium, rhodium, ruthenium, stainless steel,tungsten, titanium, nickel and alloys thererof; used singly or incombination.

The preferred size of the solid phase material depends on the type ofmaterial being used. For example, those skilled in the art willrecognize that if the solid phase is a stent or coil, the size ispreferably of a diameter that fits within a blood vessel, such as anartery. Typically the diameter will be up to about 15 mm or greater. Ifthe solid phase is a particle, such as a bead, the diameter may be up toabout 7 mm, preferably from about 1 μm to about 5 mm, even morepreferably from about 20 μm to about 300 μm. The size of solid phasematerials suitable for use in the present invention are well known tothose skilled in the art, and should not be limited to the exemplarysizes recited above.

As used herein, a binding agent or targeting moiety refers to one ormore solid phase chemical or biological molecules or structures forbinding one substance to another. Specifically the binding agent, orsolid phase agent, binds a ligand, a receptor or a ligand/receptorcomplex on a defined population of cells, typically hyperplastic tissueand/or associated vasculature, or a cancer cell and/or associatedvasculature. A molecule's function as a binding agent should not belimited by the structural mechanism of attachment. For example, abinding agent may bind a receptor, an antigenic determinant or epitope,an enzymatic substrate, or other biological structure linking thebinding agent to a target cell or cell population. The binding agent maybe a conjugate, and includes but is not limited to immunologicalconjugates, chemical conjugates (covalent or non-covalent), fusionproteins, and the like.

As used herein, a ligand-binding agent refers to a complementary set ofmolecules that demonstrate specific binding for each other. Aligand/anti-ligand pair generally binds with relatively high affinity,and for this reason, may be highly desirable for use with the presentinvention. A very well known ligand/anti-ligand pair is biotin andavidin. As used herein, avidin refers to avidin, streptavidin,neutravidin, derivatives and analogs thereof, and functional equivalentsthereof. Avidin may bind biotin in a multivalent or univalent manner.Other exemplary ligand/anti-ligand pairs include, but are not limitedto, homophyllic peptides, heterophyllic peptides, “leucine zippers”,zinc finger proteins/ds DNA fragment, enzyme/enzyme inhibitor,hapten/antibody, ligand/ligand receptor, and growth factor/growth factorreceptor.

As used herein, a selected site, a pre-determined site, targeting, andpre-targeting all refer to a site where the accumulation of plateletsabout a solid-phase agent will provide a therapeutically beneficialresult. Typically, this involves target site localization of a targetingmoiety. Such sites include, but are not limited to, the vasculature ofsolid tumors, the vasculature of benign tumors, the vasculature ofhyperplastic tissue(s), AV-malformations, vessel aneurysms andendoleaks.

As used herein, delivery of the solid agent comprising aplatelet-binding agent can occur using a catheter, a microcatheter or byneedle and syringe. Delivery by catheter or microcatheter is most oftenachieved by access through the arterial circuit, however delivery of thesolid agent through the venous circuit is also desirable. As an example,the solid agent in the form of particles, coils or stents can bedelivered by catheter to the target site using the arterial or venouscircuits. Delivery of the solid agent using the arterial circuit isadvantageous since the capillary beds downstream of the applied agent inthe target tissue act as a means of trapping the agent, therebypreventing the agent from entering the systemic circulation. The solidagent can also be localized within the arterial circulation using atargeting agent associated with the solid agent. Delivery of the solidagent using the venous system is also desirable. Localized delivery ofthe solid agent in the venous system can be accomplished by binding thesolid agent to the target site using a targeting agent associated withthe solid agent. The solid agent can also be delivered to the targetsite during a surgical procedure. As an example, the solid agent in theform of particles can be delivered by syringe and needle to the targetsite. As a further example, the solid agent in the form of a coil orstent can be placed manually at the target site during the surgicalprocedure.

As used herein, thrombus refers to any semi-solid aggregate of bloodcells enmeshed in fibrin and clumps of platelets originating fromplatelets actively binding to the solid-phase agent. In accordance withthe invention, a thrombus is formed as a direct result of activatedplatelet accumulation at the pre-determined site. Thrombosis refers tothe formation of a thrombus, typically within a blood vessel.Thrombogenic refers to substances that tend to cause thrombosis, or arethrombus forming.

As used herein, embolus refers to an intravascular mass, which travelsthrough the bloodstream, and through size constraints eventually becomeslodged in a blood vessel or capillary, distal from the site of origin ofthe intravascular mass. Embolization does not imply an active process,but instead refers to a passive process whereby occlusion of bloodvessels occurs by intravascular masses traveling through the bloodstream where they become lodged in small blood vessels and capillaries.

In contrast, the present invention involves the delivery of solid-phasematerial to target vasculature whereupon platelets are activelyrecruited to the solid-phase surface through the use of aplatelet-binding agent. In contrast to embolizing materials described incited patents, included herein as reference, the agents of the presentinvention must be delivered in close proximity to the target vasculaturedue to rapid accumulation of platelets about the solid-phase material.

The present invention improves upon existing methods of producingvascular occlusion by securing platelets to the surface of a solid-phasematerial through the use of a platelet-binding agent, thereby increasingthe effective size of the solid-phase material. For example, a particle(e.g. polylactide, polyglycolide or polylactide-co-glycolide) coatedwith or containing collagen , which is injected into the blood stream,would rapidly accumulate platelets on its surface, in effect producingan ‘onion-effect’ of layered, activated platelets in close proximity tothe injection site. Therefore the present invention enables delivery ofa minimum number of small particles into the bloodstream, whereupon theparticles rapidly grow in size from the accretion of platelets activelybinding to the platelet-binding agent on or within the particle.Furthermore, the particle-bound platelets would interact with each otherthereby forming aggregates of increasing size producing a tight matrixand effecting occlusion of the target vasculature.

The present invention further improves upon existing methods ofproducing vascular occlusion by securing platelets to the surface of asolid-phase material by means of a platelet-binding agent. The agent ofthe invention would therefore have the following effects in vivo: a)molding to the contours of the blood vessel or capillary in which itresides, b) producing a solid, impermeable three-dimensional matrix;this in turn produces a tight, impermeable seal within the vessel,thereby maximally inhibiting the delivery of blood to downstream bloodvessels and tissues.

For example, the introduction of a platelet-binding particle into theblood stream would proceed through the following sequence of events: a)a single layer of platelets would form on the surface of the particlethereby forming (i) a particle of increased diameter and (ii) a particlecoated with activated platelets with the propensity to bind and activatenearby platelets in suspension, herein defined as ‘single-layeredsurface activated platelets’ particle (S-SAP particle), b) plateletsflowing in the blood stream would interact with platelets bound to theS-SAP particle forming ‘onion-like’ layers, herein defined as‘multi-layered surface activated platelet particle (M-SAP particle), c)M-SAP would interact with each other through platelet/plateletinteraction forming larger aggregates, herein defined as the ‘M-SAPmatrix’.

As a further example, the introduction of the amorphous platelet bindingparticle polylactide, polyglycolide or polylactide-co-glycolideparticles coated with mammalian collagen into the blood stream wouldproceed through the following sequence of events: a) single plateletswould bind on and within the matrix of the particle thereby forming (i)a particle with increased diameter and rigidity, (ii) a particle coatedwith and containing activated platelets with the propensity to bind andactivate nearby platelets in suspension; b) platelets flowing in theblood stream would interact with the platelets bound to and/or boundwithin the particle thereby forming aggregates within and/or on theparticle, c) particles containing and/or having surface bound plateletswould interact with each other to form large particle aggregates.

As used herein, therapeutically beneficial, providing a therapeuticbenefit or the like refers to a desirable change in the physiology ofthe recipient animal. In a preferred embodiment of the invention, thechange is detectable. In accordance with the invention, any biologicalmechanism that involves activated platelets or platelet modulation maybe used or harnessed to achieve a beneficial therapeutic result.Exemplary therapeutic benefits produced in accordance with the presentinvention include, but are not limited to, forming a thrombus, forming aplatelet-mediated occlusion, eliminating a hyperplastic tissue or cells,eliminating a tumor and/or tumor cells, diminishing the size of ahyperplastic tissue, diminishing the size of a tumor, causing thehyperplastic tissue or tumor to become susceptible to additionaltherapies such as chemotherapy and/or radiation therapy or the like,starving or reducing the nutrient supply to a hyperplastic tissue orcancer, repairing AV-malformations, reducing or preventing blood lossfrom endoleaks and repairing vessel aneurysms.

As used herein, “administering” refers to any action that results indelivering a composition containing a solid-phase agent to apre-determined cell, cells, or tissue, typically mammalian.Administering may be conducted in vivo, in vitro, or ex vivo. Forexample, a composition may be administered by injection or through anendoscope or catheter. Administering also includes the directapplication to cells of a composition according to the presentinvention. For example, during the course of surgery, the vasculature oftumor or hyperplastic tissue may be exposed. In accordance with anembodiment of the invention, the exposed cells or vasculature may beexposed directly to a composition of the present invention, e.g., bywashing or irrigating the surgical site, vasculature, and/or the cells.

The solid-phase platelet-binding agent can be localized to a specifictarget site using a binding or targeting agent. Exemplary binding ortargeting agents include, but are not limited to: monoclonal antibodies;polyclonal antibodies; chimeric monoclonal antibodies; humanizedantibodies; genetically engineered antibodies; fragments of antibodies,selected from the group consisting of F(ab)2, F(ab′)2, Fab, F(ab′), Dab,Fv, sFv, scFv, Fc, and minimal recognition unit; single chainsrepresenting the reactive portion of monoclonal antibodies (SC-Mab);tumor-binding peptides; a protein, including receptor proteins; peptide;polypeptide; glycoprotein; lipoprotein, or the like, e.g., growthfactors; lymphokines and cytokines; enzymes, immune modulators;hormones, for example, somatostatin; a ligand (paired with itscomplementary anti-ligand); oligonucleotides; any of the above joined toa molecule that mediates an effector function; and mimics or fragmentsof any of the above. Analogs of the above-listed targeting moieties thatretain the capacity to bind to a defined target cell population may alsobe used within the claimed invention. In addition, synthetic targetingmoieties may be designed.

Monoclonal antibodies useful in the practice of the present inventioninclude whole antibodies and fragments thereof. Such monoclonalantibodies and fragments are producible in accordance with conventionaltechniques, such as hybridoma synthesis, recombinant DNA techniques andprotein synthesis. Useful monoclonal antibodies and fragments may bederived from any species (including humans) or may be formed as chimericproteins, which employ sequences from more than one species. See,generally, Kohler and Milstein, Nature, 256:495-97, 1975; Eur. J.Immunol., 6:511-19, 1976. The preferred binding and/or targeting agentcapable of localizing the solid-phase agent to a target site is anantibody or antibody-like molecule, preferably a monoclonal antibody. Amore preferred binding agent is an antibody that binds a ligand/receptorcomplex on hyperplastic tissue or cells (e.g., tumor) or the vasculatureassociated with hyperplastic tissue or cells. The most preferred bindingagent is an antibody or antibody-like molecule that binds a growthfactor/growth factor receptor complex either on or in the vicinity ofthe tumor mass such as the tumor vasculature. In a preferred embodimentof the invention, the binding agent (i.e., antibody or antibody-likemolecules) would bind to the VEGF/VEGF receptor complex. In a furtherpreferred embodiment of the invention, the antibody or antibody-likemolecule binding would recognize a neo-epitope (cryptic or previouslyunavailable epitope) formed due to ligand/receptor (i.e., growthfactor/growth factor receptor) interaction. In a further preferredembodiment of the invention, the binding of the antibody orantibody-like molecules to the growth factor/growth factor receptorcomplex would not affect the function of either the growth factor or thegrowth factor receptor.

Oligonucleotides, e.g., anti-sense oligonucleotides that arecomplementary to portions of target cell nucleic acids (DNA or RNA), arealso useful as targeting moieties in the practice of the presentinvention. Oligonucleotides binding to cell surfaces are also useful.

Functional equivalents of the aforementioned molecules are also usefulas targeting moieties of the present invention. One targeting moietyfunctional equivalent is a “mimetic” compound, an organic chemicalconstruct designed to mimic the proper configuration and/or orientationfor targeting moiety-target cell binding. Another targeting moietyfunctional equivalent is a short polypeptide designated as a “minimal”polypeptide, constructed using computer-assisted molecular modeling andmutants having altered binding affinity, such minimal polypeptidesexhibiting the binding affinity of the targeting moiety.

The Fv fragments of immunoglobulins have many significant advantagesover whole immunoglobulins for the purpose of targeted tumor therapy,including better lesion penetration on solid tumor tissue and more rapidblood clearance, as well as potentially lower Fc-mediatedimmunogenicity. An exemplary single-chain Fv (scFv) binding agent may beengineered from the genes isolated from the variable regions ofantibodies recognizing a ligand/receptor complex.

An embodiment of the invention involves a targeting agent having abinding affinity for a marker found, expressed, accessible to binding,or otherwise localized on the cell surfaces of tumor-associated vascularendothelial cells as compared to normal non-tumor-associatedvasculature. Further, certain markers for which a targeting agent has abinding affinity may be associated with components of thetumor-associated vasculature rather than on the tumor-associatedendothelial cells, themselves. For example, such markers may be locatedon basement membranes or tumor-associated connective tissue.

It may be desirable to prepare and employ an antibody or other bindingagent or moiety having a relatively high degree of selectivity for tumorvasculature, together with little or no reactivity with the cell surfaceof normal endothelial cells as assessed by immunostaining of tissuesections. It may also be desirable to prepare and employ an antibody orother binding agent or moiety capable of binding an epitope common toall vasculature.

Any composition that includes a solid-phase platelet-binding agent withor without a targeting agent according to the invention may be used toinitiate an in vivo therapeutic benefit, thrombus formation, and/or cellkilling or regression. The composition may include one or moreadjuvants, one or more carriers, one or more excipients, one or morestabilizers, one or more permeating agents (e.g., agents that modulatedmovement across a cell membrane), one or more imaging reagents, one ormore effectors; and/or physiologically-acceptable saline and buffers.Generally, adjuvants are substances mixed with an immunogen in order toelicit a more marked immune response. The composition may also includepharmaceutically acceptable carriers. Pharmaceutically acceptablecarriers include, but are not limited to, saline, sterile water,phosphate buffered saline, and the like. Other buffering agents,dispersing agents, and inert non-toxic substances suitable for deliveryto a patient may be included in the compositions of the presentinvention. The compositions may be solutions suitable foradministration, and are typically sterile, non-pyrogenic and free ofundesirable particulate matter. The compositions may be sterilized byconventional sterilization techniques.

In a preferred embodiment of the invention, a suitable compositionincludes a binding or targeting agent that binds to ligand/receptorcomplex. Exemplary antigens useful as targets in accordance with thepresent invention include, but are not limited to, antigens associatedwith cancer, including, lung, colon, rectum, breast, ovary, prostategland, head, neck, bone, immune system, blood, or any other anatomicallocation. Exemplary antigens and/or pre-determined sites include but arenot limited to VEGF/VEGF receptor complex, FGF/FGF receptor complex, orTGF.beta/TGF.beta receptor complex, p-selectin, sialyl-lewis X,endothelin, endothelin receptor, endothelin/endothelin receptor complex,alpha-fetoprotein, platelet-endothelial cell adhesion molecule (PECAM),CD31, CD34, CD36, glycoprotein Ib (GPIb), endoglin, thrombomodulin,endothelial leukocyte adhesion molecule (ELAM), intercellular adhesionmolecule 1 (ICAM-1), MHC-I, and MHC-II. The subject may be a human oranimal subject.

As noted above, a composition or method of the present inventionincludes a platelet binding agent or component. In the present inventionthe preferred platelet binding agent is collagen.

As noted above, a composition or method of the present invention mayinclude a platelet-mediated occlusion enhancer. The platelet-mediatedocclusion enhancer may be a moiety that forms a portion of abifunctional molecule as noted above, may be an ingredient in acomposition according to the invention, and/or may be administeredseparately from a composition according to the invention.

Exemplary platelet-mediated occlusion enhancers include but are notlimited to ristocetin, thrombin, heparin-induced thrombocytopenia (HIT)antibodies or portions thereof, antiphospholipid antibodies (APA) orportions thereof, whole antibody molecules via an Fc-mediated mechanism,anti-LIBS antibodies, anti-CD9 antibodies, epinephrine, thrombinreceptor activating peptide (TRAP), proteinase-activated receptor (alsoknown as protease activated receptor, PAR) agonists, cathepsin G,elastase, arachidonate, platelet activating factor (PAF), thromboxane A2(TxA2), TxA2 mimetics, phospholipase A2 (PLA2), activators of proteinkinase C (PKC), adenosine diphosphate (ADP), inducers of cyclo-oxygenase1 (COX-1), inducers of cyclo-oxygenase 2 (COX-2), collagen, vonWillebrand factor (VWF), matrix metalloproteinases (MMPs), heparin,heparan sulfate, chondroitin sulfate, ionophores, complement cascadecomponents (e.g., C5b-9) platelet microparticles, platelet membranefractions.

As noted above, a composition or method of the present invention mayinclude a platelet-mediated occlusion retarder or the like. Theplatelet-mediated occlusion retarder may be a moiety that forms aportion of a bi-functional molecule as noted above, may be an ingredientin a composition according to the invention, and/or may be administeredseparately from a composition according to the invention.

Exemplary platelet-mediated occlusion retarders include but are notlimited to aspirin, ibuprofen, acetaminophen, ketoprofen, ticlopidine,clopidogrel, indomethacin, dipyridamole, omega-3 fatty acids,prostacyclin, nitric oxide, inducers of nitric oxide, inducers of nitricoxide synthase, matrix metalloproteinase inhibitors (MMPIs, TIMPs),anti-GPIIb/IIIa agents, anti-□v□3 agents, anti-□2□1 agents, anti-CD36agents, anti-GPVI agents, aurintricarboxylic acid, thrombin receptorantagonists, thromboxane receptor antagonists, streptokinase, urokinase,tissue plasminogen activator (tPA).

In addition, it is known that platelets that have been cooled belowtheir membrane phase transition temperature (i.e., <15 degrees C.)become irreversibly activated. Although the platelets function normallyif transfused into a patient, the platelets are rapidly cleared from thebody (i.e., in approximately 24 hours, in contrast to normal circulatingplatelet life span of 7 to 10 days). Although these platelets arecleared rapidly, they bind with high avidity to PGLA/Collagen.Therefore, transfusion of cooled platelets provides an additional meansto enhance thrombus formation at the target site. Therefore, oneembodiment of the invention includes controlling platelet-mediatedocclusion by administering platelets cooled as noted above.

As noted above, the targeting moiety may be, or may be bound to, onemember of a binding pair. Methods according to the invention may requirea time period sufficient for accumulation of the targeting moiety at thesite of localization, for optimal target to non-target accumulation, foraccumulation and binding of the second member of the binding pair,and/or for clearance of unbound substances.

In accordance with the invention, two, three or more step targeting orlocalization steps may be used. Many of these protocols are well knownin the art (see, for example, U.S. Pat. No. 5,578,287 using abiotin/avidin protocol). Exemplary multiple step protocols include, butare not limited to, administering a binding agent-ligand, administeringan anti-ligand to clear unbound binding agent and to localize boundbinding agent-ligand, and administering an active agent-ligand. As usedherein, active agent refers to any therapeutic agent that is active orbecomes active and leads to a therapeutic benefit.

In accordance with a method of the invention, the binding agent must becapable of binding a ligand/receptor complex, and may be administered tothe patient by any immunologically suitable route. For example, thebinding agent may be introduced into the patient by an intravenous,intra-arterial, subcutaneous, intraperitoneal, intrathecal,intravesical, intradermal, intramuscular, or intralymphatic route. Thecomposition may be in solid, solution, tablet, aerosol, or multi-phaseformulation forms. Liposomes, long-circulating liposomes,immunoliposomes, biodegradable microspheres, micelles, or the like mayalso be used as a carrier, vehicle, or delivery system. Further more,using ex vivo procedures well known in the art, blood, plasma or serummay be removed from the patient; optionally, it may be desirable topurify the antigen in the patient's blood; the blood or serum may thenbe mixed with a composition that includes a binding agent or thesolid-phase agent according to the invention; and the treated blood orserum is returned to the patient. The clinician may compare theresponses associated with these different routes in determining the mosteffective route of administration. The invention should not be limitedto any particular method of introducing the binding agent into thepatient.

Administration may be once, more than once, or over a prolonged period.As the compositions of this invention may be used for patients in aserious disease state, i.e., life-threatening or potentiallylife-threatening, excesses of the solid-phase agent may be administeredif desirable. Actual methods and protocols for administeringpharmaceutical compositions, including dilution techniques forinjections of the present compositions, are well known or will beapparent to one skilled in the art. Some of these methods and protocolsare described in Remington's Pharmaceutical Science, Mack Publishing Co.(1982).

A solid-phase agent may be administered in combination with otherbinding agents, or may be administered in combination with othertreatment protocols or agents, e.g., chemotherapeutic agents, embolizingagents such as Gelfoam or polyvinyl alcohol (PVA) particles or the like.

As is well known in the art, a disadvantage associated withadministering treatment agents or treatment agent conjugates in vivoincludes non-target or undesirable target binding. It is therefore adesirable attribute of any administered composition to minimizenon-target binding, to minimize non-target exposure to the treatmentagent or active agent, and/or to maximize clearance of non-bound bindingagent, ligand, or active agent. Moreover, optimizing these attributestypically permits administering a higher dose of active agent, atherapeutic agent, or an element of the process that activates apreviously un-activated agent. Those skilled in the art are well versedin selecting the optimal parameters for administering the highestpossible dose while remaining safely below a toxic threshold.

In accordance with a preferred embodiment of the invention, therefore,un-activated platelets accumulate or are induced to accumulate at apre-determined site through binding to the solid-phase agent, and thenthe properly localized platelets are selectively activated.

In accordance with a preferred embodiment of the invention, activatedplatelets accumulate or are induced to accumulate at a pre-determinedsite through binding to the solid-phase agent or through platelets boundto the solid-phase agent.

The effectiveness of the present invention may be monitored byconventional assays that determine thrombus formation, morphometricstudies of thrombus formation, tumor necrosis, tumor size, tumormorphology, and/or thrombus formation that results in tumor necrosis,blood flow studies (e.g., angiography, Doppler ultrasound, radiography,CT scan, MRI), or reduction in pain symptoms. One skilled in the artwill recognize that other tests may be performed to assess or monitortherapeutic benefit.

It will be recognized by those skilled in the art that for certaincongenital and pathological conditions, some of which are listed below,it is desirable to modify a composition or method of the presentinvention to compensate for a predisposition of the patient to bleedexcessively or to thrombose. Under these circumstances, use of modifyingagents, which either enhance or dampen a method or composition of theinvention, can be employed. The use of these modifying agents ispredicted to minimize bleeding or clotting episodes. Moreover, the useof modifying agents enables controlled administration of a compositionaccording to the invention under normal circumstances (i.e., normalhemostasis).

Exemplary pro-thrombotic or pro-coagulant conditions that may warrantthe using of controllers, retarders, or agents that diminish a method orcomposition of the invention include, but are not limited to, FactorV^(Leiden) deficiency, antiphospholipid syndrome (APS), Protein C and/orProtein S and/or Antithrombin III deficiency, deep vein thrombosis(DVT), pseudo-von Willebrands disease, Type IIb von Willebrands disease,peripheral vascular disease (PVD), and high blood pressure, amongothers. Exemplary conditions that include a risk of hemorrhage that maywarrant using enhancers or agents that augment a method or compositionof the invention include but are not limited to, any condition thatincludes a risk of hemorrhage, including but not limited to coagulationfactor deficiencies, hemophilia, thrombocytopenia, and anticoagulationtherapy, among others. Controlling thrombus generation includes at leastone of altering the temperature at the pre-determined site, altering therate of blood flow at the pre-determined site, and altering the bloodpressure at the pre-determined site.

As an example of the foregoing, it will be recognized by those skilledin the art that upon initiation of the vascular occlusion process,reversal or dampening of the associated prothrombotic condition may benecessary. In such cases, administration of agents that reduce plateletreactivity will, in turn, reduce response to the vascular occlusioninitiators. Such agents are readily known by those skilled in the artand include, but are not limited to: aspirin or aspirin-like compounds,ibuprofen, acetaminophen, ketoprofen, ticlopidine, clopidogrel,indomethacin, omega-3 fatty acids, prostacyclin, nitric oxide, inducersof nitric oxide, inducers of nitric oxide synthase, matrixmetalloproteinase inhibitors (MMPIs, TIMPs), anti-GPIb agents,anti-GPIIb/IIIa agents, anti-αvβ3 agents, anti-α2β1 agents, anti-CD36agents, aurintricarboxylic acid, thrombin receptor antagonists,thromboxane receptor antagonists, streptokinase, urokinase, tissueplasminogen activator (tPA).

An exemplary process in which it may be desirable to enhance or augmentplatelet occlusion process includes thrombocytopenic (low plateletcount) patients. These individuals would benefit from concomitant orpre-administration (transfusion) of platelet products to provide anadequate resource of platelets to accomplish platelet occlusion. It willbe recognized by those skilled in the art that all transfusable productsmimicking or approximating normal platelet function can be used undersuch circumstances. Such agents include but are not limited to: randomdonor platelets, apheresis platelets, autologous platelets, washedplatelets, platelet membrane fractions, cooled platelets, frozenplatelets, particles containing or expressing platelet membranecomponents, platelet substitutes and whole blood.

As a further example, specific platelet-function enhancing agents can beemployed to boost or enhance initial platelet reactivity once targetedto the site of therapy. Agents known to those skilled in the art havebeen demonstrated to enhance existing platelet reactivity and/or lowerthe threshold limiting sufficient platelet reactivity to facilitateirreversible platelet adhesion and/or platelet degranulation and/orplatelet/platelet binding and/or platelet accretion about an existingthrombus. These agents include but are not limited to: ristocetin,thrombin, heparin-induced thrombocytopenia (HIT) antibodies or portionsthereof, antiphospholipid antibodies (APA) or portions thereof, wholeantibody molecules via an Fc-mediated mechanism, anti-ligand-inducedbinding site (anti-LIBS) antibodies or portions thereof, anti-CD9antibodies or portions thereof, epinephrine, thrombin receptoractivating peptide (TRAP), PAR agonists, cathepsin G, elastase,arachidonate, thromboxane A2 (TxA2) mimetics, TxA2, phospholipase A2(PLA2), activators of protein kinase C (PKC), adenosine diphosphate(ADP), collagen, von Willebrand factor (VWF), matrix metalloproteinases(MMPs), heparin, heparan sulfate, chondroitin sulfate, ionophores,platelet microparticles, platelet membrane fractions.

Once introduced into the bloodstream of an animal bearing a tumor,hyperplastic tissue, AV-malformation, aneurysm or endoleak, thesolid-phase agent will localize in the target vasculature; bind orimmobilize platelets, whereby immobilization activates the platelets;and the activated platelets in turn bind and activate other plateletsuntil an occlusion is formed. Platelet activation and bindingfacilitates leukocyte binding to the activated platelets furtherenhancing occlusion of the target vasculature.

EXAMPLES Example 1

The technique of preparing monoclonal antibodies against antigenic cellsurface markers is quite straightforward and may be readily carried outusing techniques well known to those of skill in the art as exemplifiedby the technique of Kohler and Milstein (1975). Generally speaking, thepreparation of monoclonal antibodies using stimulated endothelial cellsinvolves the following procedures. Cells or cell lines derived fromhuman tumors are grown in tissue culture for four or more days. Thetissue culture supernatant (“tumor-conditioned medium”) is removed fromthe tumor cell cultures and added to cultures of human umbilical veinendothelial cells (HUVEC) at a final concentration of 50% (v/v). After 2days culture, the HUVEC are harvested non-enzymatically and 1-2×10⁶cells injected intraperitoneally into mice. This process is repeatedthree times at two-week intervals, the final immunization being by theintravenous route. Three days later, the spleen cells are harvested andfused with SP2/0 myeloma cells by standard protocols (Kohler andMilstein, 1975). Hybridomas producing antibodies with the appropriatereactivity are cloned by limiting dilution.

From the resultant collection of hybridomas, one will select one or morehybridomas that produce an antibody that recognizes the activatedvascular endothelium to a greater extent than it recognizesnon-activated vascular endothelium. The ultimate goal is theidentification of antibodies having virtually no binding affinity fornormal endothelium. Suitable antibody-producing hybridomas areidentified by screening using, for example, an ELISA, RIA, IRMA, IEF, orsimilar immunoassay against one or more types of tumor-activatedendothelial cells. Once candidates have been identified, one will testfor the absence of reactivity against non-activated or “normal”endothelium or other normal tissue or cell types. In this manner,hybridomas producing antibodies having an undesirably high level ofnormal cross-reactivity for the particular application envisioned may beexcluded.

Example 2

The technique of preparing single chain antibodies that specificallyrecognize a ligand/receptor complex, specifically a growth factor/growthfactor receptor complex is employed, whereby the resulting antibodymolecules recognize the growth factor/growth factor receptor complex,but do not bind to either the growth factor or growth factor receptoralone. These antibodies can be formed through the immunization of micewith a complex of purified ligand and receptor, such as VEGF and VEGFreceptor, and the resulting V genes used to construct an antibodylibrary in filamentous phage. The phage display of antibody fragmentsallows the production of recombinant antibody molecules againstactivated endothelial cell antigens, specifically a ligand/receptorcomplex. The phage system mimics the vertebrate immune system.

Female BALB/c mice are immunized with HPLC-purified recombinant VEGF andVEGF receptor (soluble VEGF/FLT-1 receptor or VEGF/KDR receptor, asexamples) in complex in the presence of an adjuvant such as Quil A.After the appropriate antibody titre is reached (usually following thefourth boost), the mice are sacrificed and the spleens isolated.Messenger RNA (mRNA) is isolated from the spleen and transcribed tocDNA. The V genes of the cDNA are amplified and assembled as “singlechain Fv” (scFv). After digestion with the appropriate restrictionenzymes, the scFv are ligated into phagemid vectors. Competent E. colicells are then transformed with these phagemid libraries, and afterinfection with helper phage (e.g., M13K07, Pharmacia), phage particlesdisplaying the scFv are prepared. Selected clones are screened forexpression of soluble scFv binding to the ligand/receptor complex, butdo not bind to either the ligand alone or the receptor alone. Thisscreening is accomplished using standard ELISA techniques, with theligand/receptor complex, ligand and receptor used as solid-phaseantigens, respectively.

Example 3

A variety of endothelial cell markers are known that can be employed asexisting or inducible targets for the practice of this aspect of theinvention including VEGF/VPF (vascular endothelial growthfactor/vascular permeability factor), endothelial-leukocyte adhesionmolecule (ELAM-1; Bevilacqua et al., 1987); vascular cell adhesionmolecule-1 (VCAM-1; Dustin et al; 1986) intercellular adhesionmolecule-1 (ICAM-1; Osborn et al., 1989); the agent leukocyte adhesionmolecule-1 (LAM-1 agent) or even a major histocompatibility complex(MHC) Class II antigen, such as HLA-DR, HLA-DP, or HLA-DQ (Collins etal., 1984). Of these, the targeting of the VEGF/VEGF receptor complexwill likely be preferred. Monoclonal antibodies or specific peptidesrecognizing the above endothelial cell antigens can be bound to thesolid-phase agent, such as particles coated with VWF, and delivered totarget vasculature by means of a catheter or similar delivery device.The particles are thereby bound to the endothelial cells in the targetvasculature leading to platelet binding and platelet activation on theparticle, which in turn leads to platelet aggregation about the particleand eventually thrombus formation. The formed thrombus occludes thetargeted vasculature thereby preventing delivery of oxygen and nutrientsto the down-stream tissue.

Example 4

Targeting platelets to a specific site may take the form of plateletsbinding directly to the solid-phase agent through interaction VWFimmobilized on the solid-phase agent. For example, recombinant and/orhuman and/or porcine VWF immobilized on a particle of an approximatediameter of 1 μm to 5 mm can be delivered to a target site by-variousmeans, such as by catheter. Platelet binding can take place afterdelivery of the VWF-particle to the target vasculature whereby plateletsflowing in the blood stream contact the particles, bind to theparticles, spread across the particles, activate, bind other plateletsand eventually form a thrombus that occludes the blood vessel. Plateletbinding to the VWF-particle can also be initiated ex vivo, whereby theplatelets contact the particles in a vessel outside the body, and aresubsequently delivered to the target site by means of a catheter orsimilar agent delivery device. Particle size is selected such that uponinitiation of platelet reactivity with the particles (i.e., plateletbinding to the particles) progression of the particle beyond thecapillary bed cannot occur due to size limitations or because of theparticle-associated platelets and/or bound coagulation proteinsinteracting with the vessel wall receptors. VWF particle sizes,therefore, could range from about 1 μm to about 5 mm in diameter. Mostpreferably the particles will range between 5 μm and 2 mm in diameter.An even more preferable diameter of the particle would be between 20 μmand 300 μm.

Example 5 Particles

Particles of various compositions can be used as solid-phase agents forthe purpose of the present invention. The following particles have beentested for their ability to bind agents that bind platelets. Polystyrenemicrospheres were purchased from Polysciences Inc., (Warrington, Pa.)and coated with human von Willebrand factor through a passive adhesionprocess (incubation in 0.2M carbonate buffer, pH 9.0-9.6) or through acovalent linkage to derivatized beads using carbodiimide orglutaraldehyde. Several types of beads were tested including plainpolystyrene microspheres (cat. # 07310, 17134, 17135, 07312, 07313,07314), polybead amino microspheres (cat. # 19118), polybead carboxylatemicrospheres (cat. # 17141), fluoresbrite microspheres (cat. # 17155,17156), polystyrene dyed microspheres (cat. # 15715, 15714, 15716), andparamagnetic particles (cat. # 19829). All beads bound von Willebrandfactor, and upon subsequent testing bound platelets. Binding ofplatelets to the beads was confirmed by aggregometry and phase contrastmicroscopy.

Other particles tested included polyvinyl alcohol (PVA) particles (Cook,Bloomington, Ind.) and macro-aggregated albumin (MAA) particles(Edmonton Radiopharmaceutical Centre, Edmonton, Alberta, Canada). Inseparate experiments, von Willebrand factor was bound passively(carbonate buffer, as above) and covalently (glutaraldehyde linkage, asabove) to the particles. Binding of platelets to the particles was thenconfirmed using aggregometry, phase contrast microscopy and fluorescencemicroscopy (anti-CD61 antibody labeled with FITC).

Example 6 Comparison of Mammalian VWFs

Porcine VWF, bovine VWF, and human VWF were immobilized on polystyreneparticles using two approaches. The first approach (direct binding)employed passive adsorption of the material to the solid-phase particlein the presence of 0.2M carbonate (pH 9.35). The second approach(indirect binding) consisted of the isolation of VWF from porcine,bovine, and human plasmas, respectively, using an anti-VWF antibody thathad been immobilized on the surface of the solid-phase particle. In thelatter approach, the antibody (rabbit source) used was purchased fromDako (Mississauga, Ontario; cat # A0082), and as per informationprovided by the manufacturer, was confirmed to bind human, bovine andporcine von Willebrand factor. The antibody was fixed to polystyrenebeads (4.5 μm in diameter) by passive adsorption in carbonate buffer(0.2M, pH 9.35) and incubated with the respective source plasmas for 60minutes at room temperature. The beads were washed free of unboundprotein and used to challenge whole blood and platelet rich plasma fromhumans and pigs. In a like manner, human, porcine and bovine VWFs wereindividually bound directly to the beads (i.e. without a linkingantibody) by passive adsorption as outlined above, and used to challengehuman and porcine platelets (whole blood and platelet rich plasma[PRP]). In some experiments, porcine and human PRP were mixed togetherand challenged with the various agents (see below). Immobilized VWF AbSource Whole 50:50 PRP Direct Capture VWF Blood PRP (human:porcine) ✓ —Human Human Human +++++ ++++ ++++ Porcine Porcine + + — ✓ Human HumanHuman +++++ ++++ ++++ Porcine Porcine + + ✓ — Porcine Human Human ++++++++++ +++++ Porcine Porcine +++ +++ — ✓ Porcine Human Human +++++ ++++++++++ Porcine Porcine +++ +++ ✓ — Bovine Human Human + + + PorcinePorcine + + Bovine Human Human + + + Porcine Porcine + ++ weak platelet reaction,++ moderately weak platelet reaction,+++ moderate platelet reaction,++++ strong platelet reaction,+++++ very strong platelet reaction

Example 7 White Cell Interaction

Particles bound with porcine or human VWF characteristically bound humanor porcine platelets, depending on the source blood. In addition, whitecells including monocytes, granulocytes and lymphocytes were observed tointeract with the platelets bound to the particles (confirmed bydifferential staining and microscopic examination).

Platelet activation at the target site induces secondary effects thatmay enhance diminution or killing of the target tissue. Release ofagents by the activated platelets such as platelet factor 4 (PF4)inhibit angiogenesis. Post activation platelet release ofchemoattractants such as RANTES enhance the effects of leukocytes (e.g.,eosinophils, monocytes) on target tissue. Post activation expression bythe platelets of granular constituents such as CD62 will induce bindingof monocytes and polymorphonuclear leukocytes (PMNs) resulting in tissuefactor expression (monocyte; procoagulant) and cellular activation andattack (PMNs). In addition, release of CD40 ligand (CD40L) by activatedplatelets at the target site induces tissue factor expression bymonocytes leading to a local hyper-coagulable state.

Example 8

The solid-phase agent can also take the form of a coil or a stent. VWFof recombinant or mammalian origin can be bound to these solid phaseagents and delivered to the target vasculature by various meansincluding surgery and/or by catheter. The target site, such as ananeurysm, can be reached through the blood stream using specificcatheters and associated guide wires. A guide wire is introduced intothe vascular system through an entry site such as the femoral artery andgently pushed through the major blood vessels to the target site. Thecatheter is introduced over the guide wire to the target site, whereuponthe guide wire is removed. The solid-phase platelet-binding coil is thenpushed through the lumen of the catheter to the aneurysm where it isdeployed. VWF bound to the coil specifically binds platelets flowing inthe bloodstream. The platelets activate and accumulate about the coilrapidly, thereby forming a localized stationary thrombus, in turnreducing the risk of aneurismal rupture.

Example 9 Acute Effects of Particle-Immobilized VWF in a Porcine Model

The study was designed to evaluate the effectiveness of particles coatedwith human VWF in inducing thrombus formation in the vasculature of thepig kidney. In this procedure, the renal artery was catheterized using a20-22 gauge angiocatheter. The renal vessels were exposed by surgery anda Doppler flow probe attached to the renal vessels to monitor bloodflowing into and out of the target organ. No noticeable difference wasseen between flow readings taken from the renal artery and vein;therefore, all readings were further taken from the renal vein to beindicative of blood flow through the target kidney. Human VWF wascovalently bound to human MAA by overnight incubation withglutaraldehyde. Previous titration studies varying the amount of VWFbound to MAA on a per weight protein basis had established that ratiosof 1:5 through 1:80 (MAA:VWF) provided sufficient immobilized VWF toinduce platelet binding and activation. On this basis, a ratio of 1:20(MAA:VWF) was chosen for in vivo studies. Particle analysis by phasecontrast microscopy demonstrated that the particle size in the finalpreparation ranged from 30 μm through to 250 μm (as estimated by lightmicroscopy using a micrometer/hemacytometer).

Injection of MAA/VWF (500 μl or 1 ml) followed by human PRP (500 μlcontaining approximately 200—400×10⁶ platelets) into the renal artery ofthe pig caused a dramatic decrease in blood flow. Human platelets wereinjected after the test agent to approximate conditions within a humanas closely as possible. In addition, binding of porcine platelets tohuman VWF was noted to be very weak (see Example 6, above). Within 10minutes of injection of the test agent, the blood flow had dropped toless than half of baseline levels. Within 15 minutes of injection of thetest agent, the blood flow had dropped to less than 20% of the baselinevalues. Blood flow to the organ did not increase during the 90-minutemonitoring period.

Conversely, control agent (MAA alone; identical protein concentration)injected into the renal artery of the contra lateral kidney showed atransient decrease in blood flow. The blood flow decreased by 50%initially, but rapidly returned to near baseline levels, i.e., >80% oforiginal blood flow within 15 minutes and >90% of original blood flowwithin 30 minutes.

Histological examination of the target vasculature revealed a largethrombus in the immediate branches of the renal artery in the test organwith minor thrombosis of the renal vasculature in the control kidney. Nothrombi were noted in the liver, lungs, spleen, brain, heart and eyes ofthe control or test animals.

Example   Chronic Effects of Particle-Immobilized VWF in a Porcine Model

In a manner similar to the procedure outlined in Example 9, the renalarteries of two test and two control pigs were catheterized using 20-22gauge angiocatheters. A Doppler flow probe was placed surgically on therenal vein of each animal and the lead wires exposed on the outer skinof the animal after closing the initial incision. After establishing abaseline for blood flowing through the affected kidney, 1 ml of MAA:VWFparticles (11 g total protein) followed by 1 mL human platelet richplasma (approximately 460×10⁶ platelets (platelet count 458×10⁹/liter)were injected into the renal arteries of the test animals, and anidentical amount of MAA was injected into the control animals. Theanimals were transferred to metabolic crates and monitored at varyingtime intervals over a 7-day period. The following table presents bloodflow results from these studies. PORCINE DOPPLER BLOOD FLOW (millilitersper minute) Day Test 1 Control 1 Test 2 Control 2 0 28 70 35 35 1 17 1511 103 2 32 24 15 71 3 3 109 16 85 4 3 101 17 78 5 3 140 3 65 6 1 140 055 7 3 148 2 30The major organs and tissues of the control and test animals wereexamined histologically for evidence of thrombosis and othertreatment-associated damage. No thrombosis was observed except in therenal vasculature of the target kidney.

Example 11 MAA/VWF Labeling with Sodium Pertechnetate Tc 99m

MAA/VWF particles were prepared by conjugating human VWF (sourceAlphanate, Alpha Therapeutics Corp.) to MAA particles (11 mg protein;source MAA for injection, Edmonton Radiopharmaceutical Centre) usingglutaraldehyde (0.0625% v/v, final). The protein ratios ranged from 40:1to 20:1 (MAA:VWF). The functionality of the particles was checked bychallenging citrated platelet rich plasma and citrated whole blood froma healthy non-medicated (greater than 2 weeks) volunteer with theMAA/VWF particles with stirring at 500 rpm for 10 minutes (10 μlparticles+100 μl platelet source). Observation of the reaction by phasecontrast microscopy confirmed the ability of the particles to bind andactivate platelets, forming large masses of platelets and MAA/VWFparticles.

The MAA/VWF particles were then labeled with sodium pertechnetate Tc 99m using activities ranging from 88 MBq to 300 MBq. Equal volumes of theparticles in saline and sodium pertechnetate Tc 99m were incubated atroom temperature for 10 minutes. Labeling efficiency was determinedusing thin layer chromatography and found to be in excess of 95%.

The Tc 99m labeled MAA/VWF particles were then tested to evaluate theeffect of the labeling on the functionality of the particles. Phasecontrast microscopy confirmed that the Tc 99m labeled MAA/VWF particleswere capable of binding and activating platelets. There was no apparentreduction in the functional activity of the labeled particles.

Example 12 Fluoroscopy Studies of MAA/VWF Thrombosis of Porcine RenalVasculature

MAA/VWF was delivered to porcine renal vasculature using a femoralartery approach. A 5-Fr sheath catheter was inserted into the femoralartery of 18 to 20 kg piglets after general anesthesia (halothane) andguided by fluoroscopy to the left renal artery. The catheter waspositioned such that upon delivery of contrast dye, only the lower poleof the kidney's vasculature was visualized. At time zero 1 ml of MAA/VWFparticles (size range 30-250 micron) were delivered slowly (over 10seconds) to the renal vasculature. Ten (10) seconds later 1 ml human PRP(420×10⁹ per liter) was delivered and the catheter flushed with 1 mlsaline. The catheter was kept in place and after a twenty (20) minutewaiting period contrast agent was again delivered to the targetvasculature. The contrast agent was observed to pool at the end of thecatheter then move rapidly into the vasculature of the upper pole of thekidney. The vasculature of the lower pole of the kidney slowlyaccumulated contrast agent that did not dissipate (greater than 20minutes), while the upper pole vasculature rapidly lost the contrastagent (less than 5 seconds). With the animal under anesthetic and thecatheter still in place, the affected kidney was surgically exposed, therenal vasculature clamped just proximal to the catheter and the affectedkidney removed. The renal artery was opened through a longitudinalincision and dissected toward the kidney. A large thrombus was noteddistal to the tip of the catheter extending deep within the vasculatureof the lower pole of the kidney. No clotting was noted in thevasculature of the upper pole of the kidney. In addition, the lower poleof the affected kidney was noticeably blanched indicating a lack ofblood flow, while the upper pole of the kidney exhibited normal redcoloration.

In a separate experiment following the above outlined procedure thecatheter was directed to the vasculature of the lower pole of the targetkidney and used to deliver 1 ml of MAA/VWF particles followed by 1 ml ofhuman PRP. After 20 minutes blood flow to the lower pole of the kidneywas notably impeded as determined by fluoroscopy. As before, thecontrast dye moved rapidly into the upper pole vasculature after poolingat the tip of the catheter. The catheter was then repositioned andMAA/VWF followed by human PRP was delivered to the upper polevasculature. After a 20 minute waiting period contrast dye was againinjected. Fluoroscopy revealed that blood flow to the upper polevasculature was blocked. The contrast dye did not enter the lower polevasculature. The target kidney with associated renal vessels was thenremoved surgically prior to sacrificing the animal. Immediate dissectionof the kidney revealed extensive thrombosis of the blood vessels in boththe upper and lower poles of the kidney.

Example 13 Preparation of Collagen-Coated Particles

Various sources of collagen were bound to different particle types bypassive adsorption, or chemical conjugation.

Collagen derived from calf-skin and bovine tendon (Sigma; St Louis,Mo.), was bound to 4.5 μm diameter polystyrene particles (Polybead;Polysciences Corp.; Warrington, Pa.) through passive adsorption byovernight incubation in the presence of mild acid. The particles werewashed free of unbound collagen by centrifugation (3 cycles; saline,0.15 M) and left suspended in saline. The particles were stored at 4° C.until needed.

Collagen purchased from Bard Inc. as Avitene flour (Bard; Murray Hill,N.J.) was solubilized in mild acid and covalently bound to 100 μmdiameter polylactic acid (PLA) particles (G. Kisker, Germany) using EDC(Sigma) as a linking agent (3 hrs, 22° C.). Acid-solubilized Avitene wascovalently bound to 100-135 μm diameter polylactide-co-glycolide (PLGA)particles (PolyMicrospheres; Indianapolis, Ind.), or 240 μm PLGAparticles using glutaraldehyde (Fisher Scientific Inc., Edmonton,Canada) as a linking agent. Conjugations were accomplished by overnightincubation using glutaraldehyde. The particles were washed free ofunbound collagen, blocked with 1% (w/v) bovine serum albumin (BSA) andleft suspended in PBS. The particles were stored at 4° C. until needed.

Example 14 Detection of Collagen Bound to Particles

Collagen bound to the various particle types was detected using acollagen-specific monoclonal antibody. Particles were incubated withsaturating concentrations of anti-collagen IgG (Sigma) for 1 hour at 22°C. After washing away unbound antibody, the particles were incubatedwith saturating amounts of goat anti-mouse IgG-FITC for 1 hour at 22° C.(Sigma). After washing away unbound antibody, the particles wereobserved by fluorescence microscopy. Bovine serum albumin (BSA) coatedparticles served as negative control.

Example 15 Collagen-Coated Particle Mediated Platelet Activation

Collagen coated particles (acid-soluble, fibrillar, Avitene) were mixedwith citrated whole blood from healthy non-medicated donors to determinethe effect of the particles on platelet activation. Agitation of thereaction mixture was accomplished by mixing on a rotary shaker inmicrowells. Particle-dependent platelet activation was assessed by phasecontrast microscopy and by a platelet counting technique. Mixingcollagen-coated particles with whole blood induced platelet activationand binding to the particles. In contrast to the addition of acidsoluble collagen (i.e. not particle bound), which caused extensiveplatelet aggregation observed as platelet clumps in the reactionmixture, the collagen-particle-activated platelets remained localized tothe particle surface facilitating particle bound platelet aggregation.Platelet counts post-mixing were significantly less than the startingplatelet counts as determined by automated cell counting (Coulter Ac.T;Coulter, Fullerton, Calif.).

Example 16 Inhibition Studies

Citrated whole blood from healthy, unmedicated donors was incubated withsaturating concentrations of anti-GPIb antibody, clone AK2 (BiodesignInternational; Saco, Me.). The whole blood was then challenged withdecreasing numbers of collagen-coated polystyrene microspheres. BSAmicrospheres were used as negative control. The results of theexperiments are shown in the table below. Percent Platelets RemainingPercent Platelets Remaining After Reaction After Reaction (100% testagent) (1:1 dilution of test agent) Agent Donor 1 Donor 2 Donor 3 Donor1 Donor 2 Donor 3 Collagen Coated 0.93 1.1 2.39 4.97 6.29 93.99Polystyrene Microspheres Collagen Coated 1.31 5.92 34.2 23.2 75.89100.18 Polystyrene Microspheres + Anti GP1b Change in 1.4-fold 5.4-fold14.3-fold 4.7-fold 12.1-fold 1.1-fold Platelet decrease decreasedecrease decrease decrease decrease Reactivity

Anti-GPIb antibody significantly reduced platelet reactivity with thecollagen-coated microspheres indicating that the platelets wereactivated by an immobilized VWF-dependent mechanism. This result is notsurprising since collagen exposed as part of the subendothelium is knownto bind circulating VWF, which in turn serves to capture plateletsflowing past the area of vascular damage.

In a separate series of experiments, PLGA coated collagen microsphereswere used to challenge, whole blood that had been incubated withsaturating concentrations of anti-GPIb, collagen particles that had beenincubated with anti-collagen antibodies. The results of the experimentsare shown in the table below. Percent Platelets Remaining Test AgentAfter Reaction Collagen Coated PLGA particles 9.7 Collagen Coated PLGAparticles + anti collagen 90.2 antibody Whole Blood + anti GPIb antibody54.6

Both the anti-GPIb and the anti-collagen antibodies reduced plateletreactivity with the PLGA/collagen microspheres. The results demonstratethat platelets are activated in the presence of PLGA/collagenmicrospheres through two mechanisms, a VWF-dependent and aVWF-independent mechanism. Since collagen is required to capture VWFfrom the whole blood, treating the collagen-coated microspheres withanti-collagen antibody inhibited VWF binding to the collagen. Inaddition, collagen-dependent platelet activation through other receptorpathways was essentially eliminated. In contrast, inhibitingVWF/platelet interaction by blocking GPIb, had no effect on thecollagen-dependent platelet interaction. The results demonstrate thatboth pathways contribute to PLGA/collagen microsphere-dependent plateletactivation. The results also support the use of inhibitors to modulateplatelet activation induced by solid-phase platelet binding agents in anin vivo setting.

Although the present invention has been described in terms of particularpreferred embodiments, it is not limited to those embodiments.Alternative embodiments, examples, and modifications, which would stillbe encompassed by the invention, may be made by those skilled in theart, particularly in light of the foregoing teachings.

1. A method for treating a vascularized tumor or hyperplastic tissue,comprising administering to a mammal at a pre-determined site at or neara vascularized tumor or hyperplastic tissue, a solid-phase agentcomprising a binding agent comprising collagen and being capable ofbinding platelets, and subsequently inducing a thrombus in vivocomprising: binding said collagen to a solid-phase agent; capturingplatelets by exposing platelets to said collagen, inducing activation ofthe platelets, and allowing a thrombus to form at said pre-determinedsite, thereby limiting the blood supply at said pre-determined site andtreating said vascularized tumor or hyperplastic tissue.
 2. The methodof claim 1 wherein collagen is bound to a solid phase agent selectedfrom the group consisting of polystyrene beads, polylactic acidparticles, and polylactide-co-glycolide particles.
 3. The method ofclaim 1 wherein the source of collagen is a mammal.
 4. A method oftreating a vascularized tumor or hyperplastic tissue comprisingadministering to a pre-determined site at or near a vascularized tumoror hyperplastic tissue collagen bound to a solid support, allowing thecollagen to capture and activate platelets, and allowing the activatedplatelets to form a thrombus.
 5. The method of claim 4 wherein the solidsupport is selected from the group consisting of polyvinyl alcohol(PVA); polystyrene; polycarbonate; polylactide; polyglycolide;lactide-glycolide copolymers; polycaprolactone; lactide-caprolactonecopolymers; polyhydroxybutyrate; polyalkylcyanoacrylates;polyanhydrides; polyorthoesters; albumin; collagen; gelatin;polysaccharides; dextrans; starches; methyl methacrylate; methacrylicacid; hydroxylalkyl acrylates; hydroxylalkyl methacrylates; methyleneglycol dimethacrylate; acrylamide; bisacrylamide; cellulose-basedpolymers; ethylene glycol polymers and copolymers; oxyethylene andoxypropylene polymers; polyvinyl acetate; polyvinylpyrrolidone andpolyvinylpyridine; magnetic particles; fluorescent particles; animalcells; plant cells; macro-aggregated and micro-aggregated albumin;denatured protein aggregates; and liposomes; any of the above usedsingly or in combination.
 6. The method of claim 4 wherein the solidsupport is selected from polystyrene; polylactide; polyglycolide;lactide-glycolide copolymers, alone or in combination.
 7. The method ofclaim 4 wherein administering comprises administering using a catheter,microcatheter, syringe, by a surgical procedure, or by manual placement.8. The method of claim 4 further comprising administering an inhibitorthat inhibits platelets binding to the collagen.
 9. A method of treatinga vascularized tumor or hyperplastic tissue comprising administering toa pre-determined site at or near a vascularized tumor or hyperplastictissue von Willebrand Factor bound to microaggregated albumin, allowingthe VWF to capture and activate platelets, and allowing the activatedplatelets to form a thrombus.
 10. A method of treating a vascularizedtumor or hyperplastic tissue comprising administering to apre-determined site at or near a vascularized tumor or hyperplastictissue von Willebrand Factor bound to PLGA, allowing the VWF to captureand activate platelets, and allowing the activated platelets to form athrombus.
 11. The method of claim 4 further comprising administering aninhibitor that inhibits platelets binding to von Willebrand factor.